The sulfur containing adenine nucleoside derivatives S-adenosylmethionine and adenosine-5'-phosphosulfate play central roles in the metabolism of both eukaryotic and prokaryotic cells. The objectives of this research are to characterize the catalytic mechanisms and active site structures of the enzymes which catalyze the only known means of biosynthesis of these metabolic intermediates. Studies of S-adenosylmethionine (AdoMet) synthetase (ATP:L-methionine S-adenosyltransferase) from Escherichia coli will investigate the mechanism of the phosphohydrolase reaction that is part of the catalytic cycle. The means by which the hydrolytic activity is controlled so that ATP hydrolysis is coupled to AdoMet synthesis will be studied by single turnover kinetics and isotope exchange measurements. The groups which coordinate the two divalent metal ions which bind at the active site of AdoMet synthetase will be studied by measuring the effects of 170 labelled substrates and water on the electron paramagnetic resonance spectrum of enzyme-bound Mn(II). The lignads from the protein to the metal ions will be determined by labelling the metal bindings sites with the exchange-inert Cr(III) ion and characterizing proteolytic fragments containing Cr(III). The interaction of substrates and metal ion activators with the enzyme will be studied by 19F NMR of protein into which fluoroamino acids have been biosynthetically incorporated. The DNA of the cloned structural gene will be sequenced in order to determie the amino acid sequence of the enzyme. The studies of ATP sulfurylase (ATP:sulfate adenylyltrasferase) will involve cloning the structural gene of the enzyme from E. coli in order to develop a strain which produces a large amount of enzyme. Mechanistic studies will investigate the possible involvement of a covalent adenylyl-enzyme intermediate. Kinetic and isotope exchange methods will be used. The number of divalent metal ions required for catalytic activity and the groups to which these ions bind will be studied by metal ion binding measurements, kinetic studies with ATP analogs, and electron paramagnetic resonance studies of bound divalent metal ions.